Organ Cultures Of Tissue Research Articles

Cloning of the mouse endothelial selectins. Expression of both E- and P-selectin is inducible by tumor necrosis factor alpha.

E-selectin (ELAM-1) and P-selectin (GMP-140, PADGEM, CD62) have both been described as human endothelial cell adhesion molecules for neutrophils and monocytes. Cell surface appearance of these two proteins on human umbilical vein endothelial cells can be regulated by different mechanisms: E-selectin is transcriptionally induced, within hours, by tumor necrosis factor alpha (TNF-alpha) while P-selectin is transported from storage granules to the plasma membrane within minutes upon induction by various stimulating agents. We have obtained cDNA clones covering the full-length coding sequence of the homologous mouse proteins for both endothelial selectins. We show that synthesis of mouse P-selectin, like that of E-selectin, is transiently induced by TNF-alpha in several endothelioma cell lines derived from different mouse tissues. This induction was monitored on both the RNA as well as the protein level. The TNF induced, newly synthesized P-selectin protein was detected on the cell surface. Protein expression of P-selectin increased with a slightly lower rate than expression of E-selectin. In organ cultures of lung tissue from TNF-injected mice, the synthesis of P-selectin was clearly elevated compared to control mice. The bovine P-selectin homolog recognized by cross-reaction with anti-mouse P-selectin antibodies was also TNF-inducible in primary capillary endothelial cells from adrenal cortex and in aorta-derived endothelial cells. Thus, P-selectin can be regulated on two different levels. While the transport of stored P-selectin to the cell surface is controlled by regulated secretion of storage granules, the synthesis and surface expression can be induced by TNF-alpha similarly to that of E-selectin.

Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.

Expression of v-src in Embryonic Neural Retina Alters Cell Adhesion, Inhibits Histogenesis, and Prevents Induction of Glutamine Synthetase

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.

Air/liquid corneal organ culture: a light microscopic study

Air/liquid organ culture of tissues with stratified epithelial layers has been shown to encourage tight packing of cells and promote cellular differentiation. In this study human corneas cultured in a air/liquid environment were compared to paired, conventionally-cultured corneas to determine if the long-term morphology could be improved. Fourteen paired human corneas were cultured at 37 degrees C in covered culture dishes for 1 to 3 weeks. Air/liquid cultured corneas were placed epithelial-side up in a fixed position and culture medium was added to a level so that during rocking the corneal epithelia were intermittently exposed to air/liquid environments. Mate corneas were cultured using the conventional method. In this method corneas are fully submerged, epithelial-side down, in culture medium. After 3 weeks of culture significantly less epithelial intercellular edema was noted for the air/liquid cultures (p = 0.033), compared to conventional cultures. Significant improvements in cellular structure of the endothelial layers, after 1 and 3 weeks incubation (p = 0.029 and 0.000) and stromal layers, after 3 weeks in culture (p = 0.024), were also noted. We have shown that slight modifications of the organ culture environment lead to improvements in corneal morphology. Air/liquid corneal organ culture has promise for use in corneal wound healing studies and long-term culture.

Effects of Bordetella pertussis infection on human respiratory epithelium in vivo and in vitro

Bordetella pertussis infection probably involves attachment to and destruction of ciliated epithelial cells, but most previous studies have used animal tissue. During an epidemic, nasal epithelial biopsy specimens of 15 children (aged 1 month to 3 1/2 years) with whooping cough were examined for ciliary beat frequency, percent ciliation of the epithelium, and ciliary and epithelial cell ultrastructure. In addition, the in vitro effects of filtrates from a 24-h broth culture and of tracheal cytotoxin derived from B. pertussis on human nasal tissue organ culture were measured. B. pertussis was cultured from nasal swabs from 12 children. The mean ciliary beat frequency of their nasal biopsy specimens, 11.3 Hz (range, 10.4 to 13.0 Hz) was similar to that found in biopsy specimens from 10 normal children (mean, 12.5 Hz; range, 11.8 to 13.5 Hz). The abnormalities of the epithelium observed in 14 of 15 patients were a reduction in the number of ciliated cells, an increase in the number of cells with sparse ciliation, an increase in the number of dead cells, and extrusion of cells from the epithelial surface. In vitro, neither culture filtrate nor tracheal cytotoxin had any acute effect on ciliary function, but culture filtrate and tracheal cytotoxin (1 and 5 microM, respectively) caused extrusion of cells from the epithelial surface of turbinate tissue, loss of ciliated cells, an increased frequency of sparsely ciliated cells, and toxic changes in some cells. These changes were dose dependent and progressive, and between 36 and 90 h ciliary beating ceased. The observations made with patient tissue confirm that B. pertussis infection damages ciliated epithelium, and the in vitro experiments suggest that tracheal cytotoxin may be responsible for the abnormalities observed in vivo.

Growth Factor and Proto-Oncogene Expression in Psoriasis

The expression of several proto-oncogenes and growth factors was analyzed in normal skin and psoriatic lesions by RNA blot hybridization. Isolation of intact RNA from frozen biopsy samples required immediate exposure to denaturants during tissue homogenization. Lipocortin II and cyclophilin transcripts were used as internal controls. These transcripts were abundant and slightly but significantly elevated in psoriatic lesions. When results were normalized according to these reference transcripts, there was no increase in the expression of c-myc, c-Ha-ras, c-erbB (EGF receptor), c-jun, or transforming growth factor-beta (TGF-beta) transcripts in psoriatic lesions, and lesional c-fos transcripts were decreased relative to normal skin. In contrast, expression of TGF-alpha mRNA transcripts were markedly increased in psoriatic lesions even after normalization. Placement of normal or psoriatic tissue in organ culture for 2 to 4 h resulted in strong induction of c-fos, c-jun, and c-myc transcripts, but not of the other genes studied. Thus, overexpression of proto-oncogenes may be more characteristic of the epidermal response to acute injury than of the steady-state hyperplasia characteristic of psoriasis. Interferon-gamma (IFN-gamma) increased TGF-alpha mRNA levels in cultured human KC at long time intervals (24-48 h). However, of various cytokines tested, only EGF and TGF-alpha induced TGF-alpha mRNA after short time intervals (2-4 h). These results as well as the selective overabundance of TGF-alpha mRNA in psoriatic lesions among various cytokines tested suggest that activation of the EGF receptor tyrosine kinase by TGF-alpha is important in the pathogenesis of psoriatic epidermal hyperplasia.

Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes

Summary Low molecular weight polysulfated glycosaminoglycan (psgag) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to psgag stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of psgag/ml. Cells from arthritic cartilage were also more sensitive to the presence of psgag. In addition, concentrations of psgag (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of psgag inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that psgag may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by psgag, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight psgag in the treatment of equine degenerative joint disease.

REGULATION OF INTESTINAL SUCRASE EXPRESSION IN SUCKLING MOUSE INTESTINE

Intestinal disaccharidase expression in the developing post-natal animal is under a complex system of regulatory mechanisms involving pre-programmed genetic timing as influenced by circulating humoral factors, paracrine factors, and luminal nutrients. The aim of this study was to examine the relative contribution of circulating glucocorticoids and pre-programmed genetic expression of sucrase and lactase in a suckling mouse between post-natal days 13 and 15. We examined primary jejunal and ileal organ culture explants in serum-free medium at 0-2 hours and 22-24 hours of organ culture, respectively. Animals were studied at days 13, 14, and 15, with an additional group of adrenalectomy animals who underwent adrenalectomy on day 13 with organ culture at day 15. The results of sucrase activity are shown below.In summary, day 14 tissues in organ culture for 24 hours reached the same level of expression in organ culture of scrum-free media as in the intact animal on day 15. In addition, adrenalectomy on day 13 did not abolish the time-dependent expression of sucrase. However, the magnitude of expression in the basal state was diminished. We speculate that normal quantitative expression of intestinal disaccharidase in the 15-day suckling mouse is dependent upon a combination of intrinsic tuning in conjunction with circulating glucocorticoids.

Pathogenic Events During Infection of the Human Nasopharynx with Neisseria meningitidis and Haemophilus influenzae

The human nasopharynx is the natural habitat and reservoir for Neisseria meningitidis and Haemophilus influenzae type b (Hib). Meningococcal and Hib pathogenesis was studied in human nasopharyngeal tissue in organ culture. Inocula of greater than or equal to 10(6) meningococci or cfu of Hib were required for consistent production of infection in these cultures. By 24 hours meningococci and Hib grew to 10(8)-10(10) cfu/mL in culture supernatants, while 10(4)-10(7) cfu per organ culture were tissue associated. These studies further indicated that nasopharyngeal mucus contains components that specifically bind Hib; that both meningococci and Hib cause cytotoxicity, resulting in breakdown of tight junctions of epithelial cells, sloughing of ciliated cells, and ciliostasis; that pili are the most important components mediating initial attachment of meningococci to non-ciliated epithelial cells of the human nasopharynx; that Hib expresses both pilus and nonpilus adhesions that facilitate attachment to nonciliated cells; and that meningococci and Hib both invade the epithelial surface to reach the submucosa but do so by different routes. Meningococci and Hib have evolved successful, although divergent, mechanisms by which to infect the human nasopharynx.

Response of Parotid Gland Organ Culture to Radiation

Organ cultures of rhesus parotid tissue in medium enriched with homologous serum and supplemented with low levels of isoproterenol were irradiated with single photon doses of 2.5, 5.0, 7.5, 10.0, 12.5, or 15.0 Gy. Following irradiation, the tissue was incubated while being agitated for 24 h; then it was fixed in formalin. Microscopically, death of serous acinar cells was seen in areas unaltered by autolysis. Based on the numbers of nuclear aberrations, the dose response did not differ significantly from that observed at 24 h in parotid gland irradiated in vivo. The similar rapid response under the two conditions shows that apoptosis of irradiated parotid serous cells is a direct expression of interphase cell death.

Characterization of tissue from the bone-polymethylmethacrylate interface in a rat experimental model. Demonstration of collagen-degrading activity and bone-resorbing potential.

In previous studies, we described a layer of tissue that formed around methylmethacrylate cement that had been implanted into the posterior cervical spine of dogs. We are now reporting on a rat model in which we induced, in the interface between the bone of the posterior elements of the dorsal spine and methylmethacrylate, the formation of a layer of tissue that was morphologically similar to the tissue that had been produced in the dogs. As in the dogs, we noted macrophages and giant cells and we demonstrated that the interface tissue synthesized several basement-membrane components (type-IV collagen, laminin, and fibronectin). In addition, we demonstrated the synthesis of an additional extracellular-matrix protein--type-VI collagen. We also showed that extracts of organ cultures of tissue from the rat model degraded type-I collagen into three-quarter and one-quarter-length fragments. Such enzymatic activity is characterized of mammalian collagenase, an enzyme that is known to play a critical role in the resorption of bone.

Immunological and immunohistochemical analysis of rheumatoid nodules.

An attempt was made to examine the pathophysiology of the rheumatoid nodule. Significant amounts of interleukin 1-like activity and prostaglandin E2 were detected in the supernatants from in vitro organ cultures of rheumatoid nodule tissue. When fresh (but not old) rheumatoid nodules were minced and cultured in vitro prominent outgrowths of cells were observed. These cells expressed both HLA-DR and CD14 antigens but lacked conventional differentiation antigens for T cells and B cells, suggesting that they are of monocyte-macrophage origin. These data suggest that interleukin 1 and prostaglandin E2 may be deeply involved in the formation of rheumatoid nodules.

Genital virus infection and cervical neoplasia

Summary. The reported association between genital infection by herpes simplex virus (HSV) type 2 and cervical neoplasia has been investigated at a molecular level. Tissue culture of over 50 cervical cancers resulted in the development of several cell lines but none of these expressed HSV information. The permanent cell line, Cxl06, which was generated was found to contain multiple genome copies of human papillomavirus (HPV) type 16. Southern blot analysis of nine cervical cancers showed an authentic 2.8 Kb fragment of HSV-2 DNA in one of the tumours. This piece of viral DNA maps in a morphological transforming region of the HSV genome. Superinfection of cervical cancers with temperature-sensitive (ts) mutants of HSV 1 and 2 did not generate recombinant virus, suggesting that HSV DNA was not present in the tumours studied. An attempt to identify latent HSV close to the cervix in organ cultures of uterosacral ligament tissues resulted in reactivation of virus in several instances. These results are discussed against a background of decline in acceptance by many, for the HSV link with cervical cancer, and the dramatic impact of the discovery of a close relation between HPV type 16 and lower genital tract neoplasia.

Effect of different oxygen concentrations in the gas phase on biochemical activities of goat mammary tissue in organ culture

Non-secretory mammary explants from virgin goats showed higher RNA and protein synthesis in a low O2 gas phase (air) than in high O2 (95% O2). Lipid and casein synthesis was not affected significantly by the concentration of O2 in the atmosphere during culture. On the other hand, the more developed mammary tissue from primigravid goats showed higher lipid, casein and protein synthesis in 95% O2. The relative response of mammary tissue to hormones was not substantially different when cultured in the presence of a low or high O2 gas phase. As Hepes-buffered medium was found not to need a supply of CO2 to maintain the correct pH and as Hepes did not interfere with biochemical activities of cells, it is recommended to use it for cultures in a low O2 gas phase.

Role of Cyclic AMP in Proliferation of Lung Tissue in Organ Culture

The role of cyclic AMP in cell proliferation and division has been the subject of study by a number of investigators in the past 30 years, but the argument of whether cyclic AMP is a negative or a positive regulator has not been settled. We studied the effect of cyclic AMP on proliferation of normal and postpneumonectomized lung tissues in young adult rats by measuring the incorporation of tritiated thymidine into lung DNA in organ culture. In normal lung tissues the incorporation of [3H]thymidine was increased by exogenous dibutyryl cyclic AMP, or by isoproterenol or forskolin to stimulate adenylate cyclase, or by caffeine, which inhibits cAMP phosphodiesterase. The effect of isoproterenol, but not forskolin, was abolished by the beta-adrenergic blocking agent propranolol. The effect of caffeine on [3H]thymidine incorporation was further enhanced in normal lung tissues in the presence of dibutyryl cyclic AMP and in postpneumonectomized lung tissues. Imidazole, a cAMP phosphodiesterase stimulator, also increased [3H]thymidine incorporation in culture, but the effect was not magnified in the presence of exogenous dibutyryl cyclic AMP, nor in postpneumonectomized lung tissues. The data suggest that cyclic AMP acts as a positive regulator in proliferation of lung tissues.

Origin and Occurrence of Human Chorionic Gonadotropin β-Subunit Core Fragment

Human chorionic gonadotropin (hCG) beta-subunit core fragment (beta-fragment) is present in the urine of pregnant individuals as well as those with trophoblast disease and certain other cancers at concentrations 0.8 (early pregnancy) to 7 (second trimester pregnancy)-fold greater than that of hCG. The core fragment may be directly secreted by trophoblast tissue into the circulation or possibly originates from peripheral degradation of circulating hormone by the kidney. We examined the former hypothesis. We examined 24-h organ cultures of trophoblast tissue from first, second, and third trimester pregnancy. The media from this tissue contained hCG, free beta-subunit, and beta-fragment. The amount of beta-fragment present exceeded that of hCG, as was observed in second and third trimester pregnancy urine. The beta-fragment immunoreactive material produced by trophoblast tissue was compared to a standard preparation of urinary beta-fragment. The material in medium was identical to the standard beta-fragment in its elution pattern from a gel filtration column, from a reverse-phase HPLC column, from an ion-exchange gel, and from an immobilized lectin affinity column, and also by electrophoresis and immunoblotting with fragment-reactive monoclonal antibodies. We conclude that beta-fragment can also originate directly from trophoblast tissue, and could be the principal hCG beta-immunoreactive molecule secreted.

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids.

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.

The influence of Vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (Chondrocalcin)

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple sclerosis: effects of activated T-lymphocyte-derived products on organ cultures of nervous tissue

Supernatants from multiple sclerosis (MS) T-lymphocytes cause damage to both myelin and glial cells in cerebellar cultures assessed visually and by radiolabel release. Control T-lymphocytes, even after phytohaemagglutinin (PHA) stimulation, yielded supernatants which induced only slight damage, and at later times patients with other neurological diseases (OND) gave variable results. These differences suggest that MS T-lymphocytes are pre-activated in vivo to produce demyelinating factors while control T-lymphocytes are not pre-activated to the same extent. The visual evidence of activation of cerebellar macrophage-like cells was a common finding after MS T-lymphocyte supernatant treatment but there was no correlation with the severity of demyelination. There was a positive correlation between the percentage IL-2 receptor-bearing lymphocytes and the degree of supernatant-induced in vitro demyelination.

The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation.

The major apoprotein of rabbit pulmonary surfactant is a developmentally and hormonally regulated sialoglycoprotein, Mr congruent to 29,000-36,000 (SP 29-36). In the present study, specific antibodies were used to isolate cloned cDNA inserts for SP 29-36 from a fetal rabbit lung cDNA library in bacteriophage lambda gt11. Two species of cDNA of 1.9 and 3.0 kilobases (kb) in size were isolated that are complementary to two species of mRNA of 2.0 and 3.0 kb which differ primarily in the lengths of their 3'-untranslated regions. The 2.0-kb species of mRNA is approximately 5 times more abundant than the 3.0-kb mRNA. The results of Southern hybridization analysis of rabbit genomic DNA cut with a number of restriction enzymes are indicative that the two mRNA species are encoded by a single gene. The two mRNA species appear to be expressed only in rabbit lung tissue and are coordinately regulated during development in vivo and in vitro; hybridizable SP 29-36 mRNA is first detectable in rabbit lung tissue on day 26 of gestation, increases to a maximum on day 31, and declines somewhat after birth. The 3.0-kb cDNA is comprised of 57 nucleotides of 5'-untranslated region, an open reading frame of 741 nucleotides, and a 3'-untranslated region of 2,165 nucleotides that contains three poly(A)-addition signals. The most 5' of the poly(A) addition signals is utilized in synthesis of the 2.0-kb SP 29-36 mRNA, while the most 3' is utilized in synthesis of the 3.0-kb mRNA. The open reading frame of the 3.0-kb cDNA encodes a protein of 247 amino acids which is highly homologous to the major apoproteins of dog and human pulmonary surfactant. The SP 29-36 cDNAs were utilized to evaluate the effects of dibutyryl cyclic AMP (Bt2cAMP) on the levels of this mRNA in fetal rabbit lung tissue in organ culture. Bt2cAMP caused an induction of SP 29-36 mRNA that was detectable as early as 2 h after its addition to the medium. This inductive effect of Bt2cAMP was blocked when cycloheximide was also present in the medium for greater than or equal to 4 h. Cycloheximide treatment also reduced the levels of SP 29-36 mRNA in control explants; this inhibitory effect on control and Bt2cAMP-treated explants was reversed within 12 h of the removal of cycloheximide from the medium. These findings suggest that a labile protein factor mediates the transcription of the SP 29-36 gene and its induction by Bt2cAMP.